Influenza B virus (IBV) is one of the human respiratory viruses and the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find out molecular clue(s) to the increased viral pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in PB2, PB1, PA, BM2, and/or NS1 protein coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using reverse-genetically rescued viruses, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication property in the respiratory tracts of ferrets. In a subsequent mini-replicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.IMPORTANCE To investigate molecular pathogenic determinants of IBVs, which is one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and, of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein coding region. When analyzed using the reverse-genetically rescued viruses, the PA mutation appeared to exclusively contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and the replication property of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate viral polymerase complex activity pf both IBV lineages. These indicate that the PA mutation can be a newly defined, molecular pathogenic determinant of IBVs that substantiates our understanding of viral pathogenicity and public health risks of IBVs.